WO2006015534A1 - The preparation method of doxorubicin-polybutylcyanoacrylate nanoparticles - Google Patents

The preparation method of doxorubicin-polybutylcyanoacrylate nanoparticles Download PDF

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WO2006015534A1
WO2006015534A1 PCT/CN2005/001056 CN2005001056W WO2006015534A1 WO 2006015534 A1 WO2006015534 A1 WO 2006015534A1 CN 2005001056 W CN2005001056 W CN 2005001056W WO 2006015534 A1 WO2006015534 A1 WO 2006015534A1
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pbca
doxorubicin
drug
stir
dox
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Yangde Zhang
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Yangde Zhang
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    • A61K9/5138—Organic macromolecular compounds; Dendrimers obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

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  • the invention relates to a method for preparing nanometer particles for treating liver cancer.
  • liver cancer is one of the most common malignant tumors in the world. At present, the treatment of liver cancer is still clinically based on a comprehensive treatment plan, and many cases still require chemotherapy. However, the toxic side effects of chemotherapy cause loss of treatment opportunities for patients with poor liver function and general condition. In recent years, gene therapy has been produced and developed, but the premise of effective gene therapy is that nucleic acid molecules must be efficiently delivered to target cells.
  • DOX-PBCA-NP is: using HCl solution and doxorubicin hydrochloride powder to stir and dissolve, then adding DextranTM, adding n-butyl ⁇ -cyanoacrylate, stirring to be orange-yellow milk,
  • the present invention was prepared by adjusting ⁇ 2, passing through a 0.54 ⁇ filter, and drying.
  • the DOX-PBCA-NP obtained by the invention can carry a plurality of drugs including chemotherapeutic drugs and gene drugs, and the targeting performance thereof can significantly reduce the toxicity of the chemotherapeutic drugs, improve the curative effect, reduce the dosage of the drugs and the number of drugs, and improve the patient. Compliance, improve the stability of genetic drugs on ribozymes, prolong biological half-life, increase intracellular drug concentration and duration of action.
  • the invention first prepares blank PBCA nanoparticles, and then screens the factors affecting the preparation of PBCA nanoparticles encapsulating doxorubicin, and optimizes the preparation process by using the homogenization design, and further improves the drug content by improving the interaction between the model drug and the carrier.
  • the complex of doxorubicin and plasmid p53 gene was simultaneously encapsulated by the amphipathic reagent CTAB.
  • the 5% Dextran 7 was added to the micropipette.
  • the solution was added to the solution.
  • the solution was added to the solution.
  • BCA n-butyl cyanoacrylate
  • Dextran70 slowly add ⁇ -cyanoacrylate n-butyl ester (BCA) under magnetic stirring 0. 2ml in the above solution (about lOmin added), while stirring, add dropwise, close the bottle mouth, continue to stir lOOOrmp * 3h, 0, ⁇ 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 45 ⁇ ⁇ filter.
  • BCA ⁇ -cyanoacrylate n-butyl ester
  • DOX-PBCA-NP was prepared by one-step and two-step methods, and there was no significant difference in ER and LD between the two groups.
  • the ER of DOX-PBCA-NP increased with the increase of BCA monomer in the prescription, and the ER of DOX-PBCA-NP increased gradually (P ⁇ 0.05), while its LD Then it decreased with the increase of BCA monomer (P ⁇ 0.05).
  • the amount of BCA in the material increased from 0.1ml to 0.4ml.
  • the encapsulation efficiency of the nanoparticles increased from 46.59% to 96.25%, which increased by 1.07 times, and the loss of D0X decreased from 53.41% to 3.75%.
  • the drug loading amount was from 4.27%. It fell to 2.58%, only 0.53.
  • the amount of BCA monomer is increased, the particle size is also increased and the distribution is uneven. See Table 3 and Figure 3, Figure 4 and Figure 9.
  • the content of D0X - PBCA- NP drug did not change significantly with the increase of stirring speed, and there was no difference between the groups.
  • the zeta potential is negative, that is, the PBCA-NP after encapsulation of doxorubicin is still negatively charged. See Table 8 and Figure 5, Figure 6.
  • Figure 1 is a graphical representation of the effect of doxorubicin hydrochloride on DOX-PBCA-NP ER.
  • Figure 2 is a graphical representation of the effect of doxorubicin hydrochloride on D0X-PBCA-NP LD.
  • Figure 3 is a graphical representation of the effect of BCA dosage on the encapsulation efficiency of PBCA-NP doxorubicin.
  • Figure 4 is a graphical representation of the effect of BCA dosage on PBCA-NP doxorubicin drug loading.
  • Figure 5 is the relationship between the ER of DOX-PBCA-NP and its zeta potential after the addition of N3 ⁇ 4S0 4 .
  • Figure 6 shows the relationship between LD and its Zeta potential added to N3 ⁇ 4S0 ⁇ D0X-PBCA-NP.
  • Figure 7 is a two-step nanoparticle TEM (100000X).
  • Figure 8 is a one-step nanoparticle TEM (100000X).
  • Figure 10 is a prepared nanoparticle TEM (40000X).
  • Figure 11 is a prepared nanoparticle SEM (60000X).

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Abstract

A preparation method of doxorubicin-polybutylcyanoacrylate nanoparticles, whose steps are as follows: HCl solution react with doxorubicin hydrochloride powders, and stir the mixture to dissolve, followed by the addition of Dextran70, then a-butylcyanoacrylate, stir it till an orange opalescence appears, then adjust pH to 7; filter it over filter membrane of 0.45µm, and dry to give the nanoparticles. The DOX­PBCA-NP available from the invention can carry many drug including chemotherapeutics and gene drug, whose targeting property can significantly reduce the toxicity of drug, improve the therapeutic effect, reduce dose anddose frequency, improve patient compliance, increase the stability of gene drug against ribozyme, increase the drug concentration in cells and increase the action time.

Description

阿霉素一聚氰基丙烯酸正丁酯纳米粒的制备方法 技术领域  Preparation method of doxorubicin-n-butyl cyanoacrylate nanoparticle
本发明涉及一种对肝癌治疗药物纳米颗粒的制备方法。  The invention relates to a method for preparing nanometer particles for treating liver cancer.
背景技术 Background technique
. 肝癌是世界最常见的恶性肿瘤之一， 目前， 治疗肝癌临床上仍采取手术切 除为主的综合治疗方案，许多病例仍需接受化疗。但化疗的毒副反应使肝功能 及全身情况不佳者丧失治疗机会。近年来， 产生和发展了基因治疗， 但有效基 因治疗的前提是必须将核酸分子有效递送到靶细胞中。  Liver cancer is one of the most common malignant tumors in the world. At present, the treatment of liver cancer is still clinically based on a comprehensive treatment plan, and many cases still require chemotherapy. However, the toxic side effects of chemotherapy cause loss of treatment opportunities for patients with poor liver function and general condition. In recent years, gene therapy has been produced and developed, but the premise of effective gene therapy is that nucleic acid molecules must be efficiently delivered to target cells.
发明内容 Summary of the invention
本发明的目的，是要提供一种具有较强的靶向性的阿霉素一聚氰基丙烯酸 正丁酯纳米粒 （DOX PBCA-NP) 的制备方法。  It is an object of the present invention to provide a method for preparing doxorubicin-n-butyl cyanoacrylate nanoparticles (DOX PBCA-NP) having a strong targeting property.
本发明 DOX- PBCA-NP的制备方法是：用 HC1溶液与盐酸阿霉素粉末作用搅拌 使其溶解， 再加入 Dextran™, 加入 α—氰基丙烯酸正丁酯， 搅拌待出现橙黄色 乳光， 调节 ΡΗ二 7, 过 0. 45 μ ηι滤膜， 干燥， 即制得本发明。  The preparation method of the present invention DOX-PBCA-NP is: using HCl solution and doxorubicin hydrochloride powder to stir and dissolve, then adding DextranTM, adding n-butyl α-cyanoacrylate, stirring to be orange-yellow milk, The present invention was prepared by adjusting ΡΗ2, passing through a 0.54 μηη filter, and drying.
用本发明获得的 DOX- PBCA-NP, 可运载包括化疗药物、 基因药物在内的多 种药物， 其靶向性能显著降低化疗药物的毒性， 提高疗效， 能减少药物用量及 用药次数，提高患者依从性，提高基因药物对核酶的稳定性，延长生物半衰期， 提高胞内药物浓度和作用时间。  The DOX-PBCA-NP obtained by the invention can carry a plurality of drugs including chemotherapeutic drugs and gene drugs, and the targeting performance thereof can significantly reduce the toxicity of the chemotherapeutic drugs, improve the curative effect, reduce the dosage of the drugs and the number of drugs, and improve the patient. Compliance, improve the stability of genetic drugs on ribozymes, prolong biological half-life, increase intracellular drug concentration and duration of action.
本发明先制备出空白 PBCA纳米粒，然后筛选影响包封阿霉素的 PBCA纳米粒 制备的诸因素，采用均勾设计优化制备工艺， 从改善模型药物与载体的相互作 用进一步提高其药物含量， 并通过双亲性试剂 CTAB同时包封阿霉素和质粒 p53 基因的复合物。  The invention first prepares blank PBCA nanoparticles, and then screens the factors affecting the preparation of PBCA nanoparticles encapsulating doxorubicin, and optimizes the preparation process by using the homogenization design, and further improves the drug content by improving the interaction between the model drug and the carrier. The complex of doxorubicin and plasmid p53 gene was simultaneously encapsulated by the amphipathic reagent CTAB.
单因素初选  Single factor primary selection
1、 空白 PBCA— NP的制备  1. Preparation of blank PBCA-NP
移液管移取 0. IN HC1溶液加入锥形瓶中， 微量移液器加入 1. 5%DeXtran7。， 磁力搅拌下缓慢加入 α—氰基丙烯酸正丁酯（BCA) 0. 2ml于上述溶液（约 lOmin 加完） ， 边滴加边搅拌， 滴加完毕， 封闭瓶口， 持续搅拌 1000rpm*3h， 体系出 现白色乳光时终止反应， 0. 2N NA0H溶液调节 1¾=7. 0， 过 0. 45 μ πι滤头。 取少 量滤液于真空干燥箱中 25° (：、一 0. lkPa条件下干燥，样品送电镜室观察粒径粒 形， 余备用。 5% De X tran 7。 Pipette pipette 0. IN HC1 solution was added to the Erlenmeyer flask, the micropipette was added to 1. 5% De X tran 7 . Slowly add n-butyl cyanoacrylate (BCA) under magnetic stirring. 0. 2ml in the above solution (about 10 min addition), while stirring, add dropwise, add the bottle, and keep stirring for 1000 rpm*3h. System out The reaction was terminated in the presence of white opalescence, and the 0. 2N NA0H solution was adjusted to 13⁄4 = 7. 0, over 0. 45 μ πι filter. A small amount of the filtrate was dried in a vacuum drying oven at 25 ° (:, a 0. lkPa condition, and the sample was sent to a mirror room to observe the particle size and shape, and the remaining was used.
2、 不同制备工艺对 DOX- PBCA- NP药物含量的影响。  2. The effect of different preparation processes on the drug content of DOX-PBCA-NP.
2. 1 一步法  2. 1 one-step method
移液管移取 0. 1N HC1溶液加入锥形瓶中，取干燥至恒重的盐酸阿霉素粉末 10. OOmg于上述溶液， 搅拌至溶，微量移液器加入 1. 5%Dextran7。， 磁力搅拌下 缓慢加入 α—氰基丙烯酸正丁酯 （BCA) 0. 2ml于上述溶液 （约 lOmin加完） ， 边滴边搅拌， 滴加完毕， 封闭瓶口， 持续搅拌 1000rmp*3h， 体系出现橙黄色乳 光时终止反应， 0. 2N NaOH溶液调节 PH=7. 0， 过 0. 45 μ πι滤头。 The 5% Dextran 7 was added to the micropipette. The solution was added to the solution. The solution was added to the solution. Slowly add n-butyl cyanoacrylate (BCA) under magnetic stirring. 0. 2ml in the above solution (about 10 minutes after the addition), while stirring, add dropwise, close the bottle mouth, continue to stir 1000rmp*3h, system The reaction is terminated when the orange-yellow emulsion is present, and the pH of the solution is adjusted to 0. 2N NaOH solution.
2. 2二步法  2. 2 two-step method
移液管移取 0. IN HC1溶液加入锥形瓶中， 微量移液器加入 1. 5%  Pipette removal 0. IN HC1 solution was added to the Erlenmeyer flask, and the micropipette was added 1. 5%
Dextran70, 磁力搅拌下缓慢加入 α—氰基丙烯酸正丁酯 （BCA) 0. 2ml于上述 溶液（约 lOmin加完） ， 边滴边搅拌， 滴加完毕， 封闭瓶口， 持续搅拌 lOOOrmp *3h，体系出现白色乳光时终止反应，取干燥至恒重的盐酸阿霉素粉末 10. OOmg 于上述溶液， 搅拌至溶， 继续搅拌 2h, 0. 2N NaOH溶液调节 PH=7. 0， 过 0. 45 μ πι 滤头。 Dextran70, slowly add α-cyanoacrylate n-butyl ester (BCA) under magnetic stirring 0. 2ml in the above solution (about lOmin added), while stirring, add dropwise, close the bottle mouth, continue to stir lOOOrmp * 3h, 0,过0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 45 μ πι filter.
各种相关因素对药物含量的影响：  The impact of various relevant factors on the drug content:
1、 制备方法对 DOX- PBCA-NP药物含量的影响  1. Effect of preparation methods on drug content of DOX-PBCA-NP
采用一步法和二步法制备 DOX- PBCA-NP, 两组间 ER和 LD差异无显著性 DOX-PBCA-NP was prepared by one-step and two-step methods, and there was no significant difference in ER and LD between the two groups.
(Ρ>0. 05) ， 见表 1， 但一步法制备的纳米粒粒径较二步法大。 如图 7、 图 8。 (Ρ>0. 05), see Table 1, but the particle size of the nanoparticle prepared by one-step method is larger than that of the two-step method. Figure 7, Figure 8.
制备工艺对 DOX- PBCA- NP ER和 LD的影响 （η=3)  Effect of preparation process on DOX- PBCA- NP ER and LD (η=3)
制备工艺 ER(%) LD (%)  Preparation Process ER(%) LD (%)
一步法 65. 22+/- 1. 64 3. 46+/- 0. 09  One-step method 65. 22+/- 1. 64 3. 46+/- 0. 09
二步法 63. 54+/-0. 76# 3. 42+/- 0. 04# Two-step method 63. 54+/-0. 76 # 3. 42+/- 0. 04 #
注： #相邻两组间相比 P〉0. 05。  Note: # Comparable between the two groups P>0.05.
2、 盐酸阿霉素用量对 D0X-PBCA-NP药物含量的影响 依 2.1处方和工艺， DOX-PBCA- NP的 ER随着处方中的阿霉素的用量的增加而 逐渐降低， 差异有显著性（P<0.05或 P<0.01) ， 而其载药量则随着处方中阿霉 素的增加而增加， 5.0mg组低于其它各组 (P<0.05) ， 但 20. Omg组与 15. Omg组 的载药量无显著性差异 （P〉0.05) 。 如表 2和图 1、 图 2。 2, the effect of doxorubicin hydrochloride on the drug content of D0X-PBCA-NP According to the 2.1 prescription and process, the ER of DOX-PBCA-NP gradually decreased with the increase of the dosage of doxorubicin in the prescription, the difference was significant (P<0.05 or P<0.01), and the drug loading amount was The increase of doxorubicin in the prescription increased, and the 5.0 mg group was lower than the other groups (P<0.05), but there was no significant difference in the drug loading between the 20. Omg group and the 15. Omg group (P>0.05). See Table 2 and Figure 1, Figure 2.
表 2 盐酸阿霉素用量对 DOX-PBCA-NP药物含量的影响 （n=3)  Table 2 Effect of Doxorubicin Hydrochloride on Drug Content of DOX-PBCA-NP (n=3)
DOX量 （mg) ER(%)
Figure imgf000005_0001
DOX amount (mg) ER (%)
Figure imgf000005_0001
5.0 76.80+/-0.02 2.11+/—0.05  5.0 76.80+/-0.02 2.11+/-0.05
10.0 65.22+/- 1.64* 3.46+/- 0, 09*  10.0 65.22+/- 1.64* 3.46+/- 0, 09*
15.0 44.89+/- 0.36* 3.70+/- 0.03*  15.0 44.89+/- 0.36* 3.70+/- 0.03*
20.0 33· 76+/-0.46* 3.71+/-0.05# 20.0 33· 76+/-0.46* 3.71+/-0.05 #
注： *表示相邻组相比 Ρ<0.05， #表示相邻两组间相比 Ρ>0.05。  Note: * indicates that the adjacent group is Ρ<0.05, and # indicates Ρ>0.05 compared between the two groups.
3、 BCA单体用量对 DOX- PBCA-NP药物用量的影响  3. Effect of BCA monomer dosage on DOX-PBCA-NP dosage
参照 2.1工艺，改变处方中 BCA单体的用量后， DOX-PBCA-NP的 ER随着处方中 的 BCA单体的增加， DOX- PBCA-NP的 ER逐渐增加 （P〈0.05) ，而其 LD则随 BCA单体 的增加而降低（P<0.05) 。材料 BCA量从 0.1ml增至 0.4ml， 纳米粒的包封率相应 从 46.59%增至 96.25%， 增加 1.07倍， D0X的损耗则由 53.41%降至 3.75%; 而 载药量则从 4.27%降至 2.58%, 仅为原来的 0.53。 BCA单体用量增加的同时其 粒径也增大， 分布不均。 如表 3和图 3、 图 4及图 9。  According to the 2.1 process, after changing the dosage of BCA monomer in the prescription, the ER of DOX-PBCA-NP increased with the increase of BCA monomer in the prescription, and the ER of DOX-PBCA-NP increased gradually (P<0.05), while its LD Then it decreased with the increase of BCA monomer (P<0.05). The amount of BCA in the material increased from 0.1ml to 0.4ml. The encapsulation efficiency of the nanoparticles increased from 46.59% to 96.25%, which increased by 1.07 times, and the loss of D0X decreased from 53.41% to 3.75%. The drug loading amount was from 4.27%. It fell to 2.58%, only 0.53. When the amount of BCA monomer is increased, the particle size is also increased and the distribution is uneven. See Table 3 and Figure 3, Figure 4 and Figure 9.
表 3 BCA单体用量对 DOX- PBCA-NP药物含量的影响 （n=3)  Table 3 Effect of BCA monomer dosage on DOX-PBCA-NP drug content (n=3)
BCA量 （ml) ER(%) LD(%)  BCA amount (ml) ER (%) LD (%)
0.1 46.59+/— 1.28 4.87+/— 0.14  0.1 46.59+/- 1.28 4.87+/— 0.14
0.2 65.22+/- 1.64* 3.46+/- 0.09*  0.2 65.22+/- 1.64* 3.46+/- 0.09*
0.3 87.05+/-2.46* 3· 09+/- 0.07*  0.3 87.05+/-2.46* 3· 09+/- 0.07*
0.4 96.25+/- 0.38* 2.58+/- 0.01*  0.4 96.25+/- 0.38* 2.58+/- 0.01*
注： *表示相邻组相比 Ρ<0.05  Note: * indicates that the adjacent group is Ρ<0.05
4、 值对 DOX-PBCA- NP药物含量的影响  4, the value of the effect of DOX-PBCA- NP drug content
BCA单体 0.2ml， D0X10. Omg, 依 2.1工艺制备 DOX- PBCA-NP, ER随着 PH值的 增大而逐渐减小， 但 PH=2， 3两组的 ER与 PH二 1时的 ER相比， 差异无显著性0.2% of BCA monomer, D0X10. Omg, DOX-PBCA-NP prepared according to 2.1 process, ER with PH value Increased and gradually decreased, but PH=2, 3 ER compared with ER at pH 2, the difference was not significant
(P>0. 05 ) ， 当 PH值增至 5以上时， ER明显减小， PH=5， 6组与 PH=1组相比差异 有显著性 (P<0. 05) ， 载药量在 PH二 2， 3， 5时虽然减小， 但与 PH=1时相比差异 不显著 （P>0. 05 ) 。 见表 4。 另外， 体系出现乳光的时间随着 PH值的降低而逐 渐延长， PH=1时反应时间长达 6- 7小时。 还发现当体系 PH值〉 8时制备的纳米粒 难以成球， 产品在镜下呈片状物， 见图 10。 (P>0.05), when the PH value increased to 5 or more, ER decreased significantly, PH=5, and the difference between the 6 groups and the PH=1 group was significant (P<0.05), drug loading. Although the decrease was at PH 2, 3, and 5, the difference was not significant compared with PH = 1 (P > 0.05). See Table 4. In addition, the time for the opalescence of the system gradually increases with the decrease of the PH value, and the reaction time is as long as 6-7 hours at pH=1. It has also been found that when the pH value of the system is > 8, the nanoparticles prepared are difficult to form a sphere, and the product is in the form of a sheet under the microscope, as shown in Fig. 10.
表 4 PH值对 D0X- PBCA-NP药物含量的影响 （n = 3 ) Table 4 Effect of PH on D0X-PBCA-NP drug content (n = 3)
Figure imgf000006_0001
Figure imgf000006_0001
1. 0 65. 42+/- 3. 10 3. 46+/- 0. 17  1. 0 65. 42+/- 3. 10 3. 46+/- 0. 17
2. 0 65. 22+/-1. 64 3. 46+/- 0. 09  2. 0 65. 22+/-1. 64 3. 46+/- 0. 09
3. 0 65. 22+/— 2. 00 3. 46+/-0. 11  3. 0 65. 22+/- 2. 00 3. 46+/-0. 11
5. 0 63. 86+/- 3. 65* 3. 39+/- 0. 20  5. 0 63. 86+/- 3. 65* 3. 39+/- 0. 20
6. 0 54. 74+/-4. 38* 2. 92+/- 0. 24*  6. 0 54. 74+/-4. 38* 2. 92+/- 0. 24*
注： *表示与 PH=1. 0组相比 P〈0. 05  Note: * indicates that compared with PH=1. 0 group P<0.05
5、 搅拌速度对 DOX- PBCA-NP药物含量的影响  5, the effect of stirring speed on the drug content of DOX- PBCA-NP
D0X - PBCA- NP药物含量随着搅拌速度的增加而变化不明显， 各组间无差异 The content of D0X - PBCA- NP drug did not change significantly with the increase of stirring speed, and there was no difference between the groups.
(P>0. 05 ) ，唯粒经分布随着搅拌速度增大而增宽。 见表 5 (P>0.05), only the grain distribution increases as the stirring speed increases. See Table 5
表 5 搅拌速对 DOX- PBCA- NP药物含量的影响 （n=3 )  Table 5 Effect of stirring speed on DOX-PBCA-NP drug content (n=3)
搅拌速度 （rpm) ER (%) LD (%)  Stirring speed (rpm) ER (%) LD (%)
1000 65. 22+/- 1. 64 3. 46+/-0. 09  1000 65. 22+/- 1. 64 3. 46+/-0. 09
2000 65. 29+/-1. 26# 3. 46+/-0. 06# 2000 65. 29+/-1. 26 # 3. 46+/-0. 06 #
3000 65. 31+/- 0. 78# 3. 47+/- 0. 04* 3000 65. 31+/- 0. 78 # 3. 47+/- 0. 04*
注： #表示与搅拌速度 = 1000rpm组相比 P>0. 05。  Note: # indicates a comparison with the stirring speed = 1000 rpm group P>0.05.
6、 均匀设计安排结果  6, uniform design arrangement results
依均匀设计方案实验， 电镜下评估纳米粒外观（粒径、 粒形） ， 分光光度 法测定阿霉素包封率， 计算载药量， 结果如表 6。 经多元回归分析处理， 得多 元线性回归方程： Y=19. 456+0. 113¾-1. 66 - 6. 121¾- 0. 839 ， F二 0. 888, R-0. 686, R2二 0. 57, Ρ二 0. 544。 由方程可见， ¾、 、 系数均为负值， 表明 其取值宜小， 而 系数为正值， 表明其取值宜适当的大。 结合单因素分析得结 果， PH=1时反应太慢，粒径分布较宽，综合考虑后的优化条件为： DOX-10. Omg， BCA单体 =0.25ml， PH=2.5, 稳定剂 = 1.5%， 按上述条件制备纳米粒， 结果包 封率为 79.31+/- 1.17%， 载药量为 3.49+/- 0.05%， 外观如图 11。 According to the uniform design experiment, the appearance of the nanoparticles (particle size, grain shape) was evaluated under electron microscope. The encapsulation efficiency of doxorubicin was determined by spectrophotometry, and the drug loading was calculated. The results are shown in Table 6. After multiple regression analysis, the multiple linear regression equation is obtained: Y=19. 456+0. 1133⁄4-1. 66 - 6. 1213⁄4- 0. 839 , F two 0. 888, R-0. 686, R 2 2 57, Ρ 2, 0. 544. It can be seen from the equation that the 3⁄4, and the coefficients are all negative, indicating The value should be small, and the coefficient is positive, indicating that the value should be appropriate. Combined with single factor analysis, the reaction is too slow and the particle size distribution is wide at pH=1. The optimal conditions after comprehensive consideration are: DOX-10. Omg, BCA monomer = 0.25ml, PH=2.5, stabilizer = 1.5 %, the nanoparticles were prepared according to the above conditions, and the encapsulation efficiency was 79.31 +/- 1.17%, and the drug loading was 3.49 +/- 0.05%. The appearance is shown in Fig. 11.
表 6 均匀设计的实验结果评分 Table 6 Evaluation of the experimental results of uniform design
Figure imgf000007_0001
Figure imgf000007_0001
7.5 8.21 0.148 15.858  7.5 8.21 0.148 15.858
4.0 5.73 0.087 10.817  4.0 5.73 0.087 10.817
7.0 7.55 0.318 14.686  7.0 7.55 0.318 14.686
3.0 3.24 0.092 6.332  3.0 3.24 0.092 6.332
7.5 5.49 0.418 13.408  7.5 5.49 0.418 13.408
8.6 8.26 0.389 17.249  8.6 8.26 0.389 17.249
7.8 4.07 0.763 12.733  7.8 4.07 0.763 12.733
7.5 4.31 0.317 14.947  7.5 4.31 0.317 14.947
4.0 1.98 0.103 6.083  4.0 1.98 0.103 6.083
7.电解质对 DOX- PBCA-NP药物含量的影响  7. Effect of electrolyte on the drug content of DOX- PBCA-NP
随着处方中三种电解质加入， 各试验组的 D0X药物含量均有所增加， 但只 有硫酸钠组与对照组相比包封率和载药量增加的差异有显著性 （P〈0.05) ，而 NaCl, N C03组与对照组相比药物含量增加的差异性不显著（P>0.05)。如表 7。 实验过程中还发现，体系中加入上述剂量的电解质后，各实验组与对照组均未 见明显的肉眼观絮凝和聚结现象发生。 With the addition of three electrolytes in the prescription, the D0X drug content of each test group increased, but only the difference between the encapsulation efficiency and the drug loading was significantly higher in the sodium sulfate group than in the control group (P<0.05). There was no significant difference in the increase of drug content between NaCl and N C0 3 groups compared with the control group (P>0.05). As shown in Table 7. During the experiment, it was also found that after adding the above-mentioned electrolytes in the system, no obvious macroscopic flocculation and coalescence occurred in each experimental group and the control group.
表 7 电解质对 DOX- PBCA- NP药物含量的影响 （n=3)  Table 7 Effect of electrolyte on DOX-PBCA-NP drug content (n=3)
电解质 ER(%) LD(%)  Electrolyte ER(%) LD(%)
none 82.31+/- 1.03 3.49+/- 0.05  None 82.31+/- 1.03 3.49+/- 0.05
NaCl 82.70+/- 0.90 3.50+/-0.04  NaCl 82.70+/- 0.90 3.50+/-0.04
Na2C03 83.83+/-0.34 3.55+/-0.01 Na 2 C0 3 83.83+/-0.34 3.55+/-0.01
N¾S04 84.26+/-0.27* 3.57+/- 0.01* N3⁄4S0 4 84.26+/-0.27* 3.57+/- 0.01*
注： *表示与对照组相比 P〈0.05  Note: * indicates that compared with the control group, P < 0.05
8.不同浓度的 Na2S0对 D0X-PBCA-NP药物含量的影响 加入 Na2S04后， DOX- PBCA- NP的 ER和 LD增加， 8.0， 12· Omg/ml二个剂量水平 组包封率和载药量均较未加 N S04组增高， 差异有显著性（P〈0.05) ， 但 N S04 达 16. Omg/ml时药物含量较对照组并未增加，差异无显著性 (P〉0.05), 4. Omg/ml 水平组与对照组相比差异无显著性（P>0.05)，对应的 Zeta电位亦有相同改变。 N S04的剂量水平在 0mg/ml〜16. Omg/ml时，纳米粒的 Zeta电位的大小与 ER和 LD 呈高度的负相关 （rER=0.9671， rLD=0.9125, P<0.05) 。 其 Zeta电位均为负值， 即包封阿霉素后的 PBCA- NP仍然带负电。 如表 8和图 5、 图 6。 8.Effects of different concentrations of Na 2 S0 on D0X-PBCA-NP drug content After adding Na 2 S0 4 , the ER and LD of DOX-PBCA-NP increased, and the encapsulation efficiency and drug loading of the two dose levels of 8.0, 12·Omg/ml were higher than those of the non-N S0 4 group. The difference was significant. Sexuality (P<0.05), but the drug content of N S0 4 reached 16. Omg/ml did not increase compared with the control group, the difference was not significant (P>0.05), 4. Omg/ml level group compared with the control group There was no significant difference (P>0.05), and the corresponding zeta potential also had the same change. When the dose level of N S0 4 was from 0 mg/ml to 16.0 mg/ml, the zeta potential of the nanoparticles was highly negatively correlated with ER and LD (r ER = 0.9671, r LD = 0.9125, P < 0.05). The zeta potential is negative, that is, the PBCA-NP after encapsulation of doxorubicin is still negatively charged. See Table 8 and Figure 5, Figure 6.
表 8 不同浓度的 N¾S0^ D0X- PBCA-NP药物含量的影响 （n=3)  Table 8 Effects of different concentrations of N3⁄4S0^ D0X- PBCA-NP on drug content (n=3)
Na2S04 (mg/ ½1) 0 4.0 8.0 12.0 16.0 Na 2 S0 4 (mg/ 1⁄21) 0 4.0 8.0 12.0 16.0
ER(%) 79.31+/-1.17 83.11+/-1.12 83.90+/- 3.03* 84.25+/- 4.19* 80.00+/- 2.11 ER (%) 79.31+/-1.17 83.11+/-1.12 83.90+/- 3.03* 84.25+/- 4.19* 80.00+/- 2.11
LD(%) 3.49+/- 0.05 3.53+/- 0.04 3.56+/- 0.13* 3.57+/-0.18* 3.40+/- 0.09LD(%) 3.49+/- 0.05 3.53+/- 0.04 3.56+/- 0.13* 3.57+/-0.18* 3.40+/- 0.09
Zeta (mv) -18.3+/-0.9 -26.7+/- 1.2 -31.4+/-1.8* -34.5+/-1.3* -16.6+/-1.7 注： *表示与对照组相比 P<0.05 Zeta (mv) -18.3+/-0.9 -26.7+/- 1.2 -31.4+/-1.8* -34.5+/-1.3* -16.6+/-1.7 Note: * indicates P<0.05 compared with the control group
9、 离子对试剂对 DOX- PBCA- NP药物含量的影响  9. Effect of ion pair reagent on DOX-PBCA-NP drug content
随着含羧基 C00-化合物油酸钠的加入， 6.0， 9. Omg/ml组与对照组相比包 封率、 载药量增加， 差异有显著性（P〈0.05) ， 但 3. Omg/ml组与对照组相比差 异无显著性 （P>0.05) 。 见表 9  With the addition of sodium carboxy-containing C00-compound oleate, the encapsulation efficiency and drug loading were significantly higher in the 6.0, 9. Omg/ml group compared with the control group (P<0.05), but 3. Omg/ There was no significant difference between the ml group and the control group (P>0.05). See Table 9
表 9 离子对试剂对 DOX- PBCA- NP药物含量的影响 （n二 3)  Table 9 Effect of ion-pairing reagent on DOX-PBCA-NP drug content (n 2 3)
油酸钠（mg/ml) 0 3.0 6.0 9.0 Sodium oleate (mg/ml) 0 3.0 6.0 9.0
ER(%) 79.31+/ - 1.17 82.70+/ - 3.32 85.68+/-1.40* 87. Θ8+/-2.55* ER(%) 79.31+/ - 1.17 82.70+/ - 3.32 85.68+/-1.40* 87. Θ8+/-2.55*
LD(%) 3.49+/-0.05 3.59+/-0.14 3.63+/- 0.06* 3.71+/-0.11* 注: *表示与对照组相比 P<0.05 LD(%) 3.49+/-0.05 3.59+/-0.14 3.63+/- 0.06* 3.71+/-0.11* Note: * indicates P<0.05 compared with the control group
10、 p53- DOX- PBCA-纳米粒的药物含量  10. Drug content of p53- DOX- PBCA-nanoparticles
加入季胺盐 CTAB后， p53的包封率明显提高， 差异有显著性（P〈0.05)，而阿 霉素的包封率变化不大， 与对照组比较差异无显著性 （P〉0.05) 。 见表 10 表 10 p53- DOX- PBCA-纳米粒的包封率 （n=3) After the addition of quaternary ammonium salt CTAB, the encapsulation efficiency of p53 was significantly increased (P<0.05), while the encapsulation efficiency of doxorubicin was not changed significantly, and there was no significant difference compared with the control group (P>0.05). . See Table 10 Table 10 Encapsulation efficiency of p53- DOX- PBCA-nanoparticles (n=3)
CTAB量 （ μ M) 0 500.0  CTAB amount ( μ M) 0 500.0
DOX 82.33+/- 0.09 82.17+/-0.22  DOX 82.33+/- 0.09 82.17+/-0.22
ER(%)  ER (%)
p53 23.86+/- 0.28 49.67+/-1.26*  P53 23.86+/- 0.28 49.67+/-1.26*
注： *表示与对照组相比 P<0.05 Note: * indicates P<0.05 compared with the control group.
附图说明 DRAWINGS
图 1是盐酸阿霉素用量对 DOX-PBCA-NP ER的影响图示。  Figure 1 is a graphical representation of the effect of doxorubicin hydrochloride on DOX-PBCA-NP ER.
图 2是盐酸阿霉素用量对 D0X-PBCA- NP LD的影响图示。  Figure 2 is a graphical representation of the effect of doxorubicin hydrochloride on D0X-PBCA-NP LD.
图 3是 BCA用量对 PBCA- NP阿霉素包封率的影响图示。  Figure 3 is a graphical representation of the effect of BCA dosage on the encapsulation efficiency of PBCA-NP doxorubicin.
图 4是 BCA用量对 PBCA-NP阿霉素载药量的影响图示。  Figure 4 is a graphical representation of the effect of BCA dosage on PBCA-NP doxorubicin drug loading.
图 5是加入 N¾S04后 DOX- PBCA-NP的 ER与其 Zeta电位的关系。 Figure 5 is the relationship between the ER of DOX-PBCA-NP and its zeta potential after the addition of N3⁄4S0 4 .
图 6是加入 N¾S0^D0X- PBCA-NP的 LD与其 Zeta电位的关系。  Figure 6 shows the relationship between LD and its Zeta potential added to N3⁄4S0^D0X-PBCA-NP.
图 7是二步法所得纳米粒 TEM (100000X) 。  Figure 7 is a two-step nanoparticle TEM (100000X).
图 8是一步法所得纳米粒 TEM (100000X) 。  Figure 8 is a one-step nanoparticle TEM (100000X).
图 9是 BCA单体 =0.60ml时所得纳米粒 TEM (35000X) 。  Figure 9 shows the obtained nanoparticle TEM (35000X) at BCA monomer = 0.60 ml.
图 10是制备的纳米粒 TEM (40000X) 。  Figure 10 is a prepared nanoparticle TEM (40000X).
图 11是制备的纳米粒 SEM (60000X) 。  Figure 11 is a prepared nanoparticle SEM (60000X).
具体实施方式 detailed description
下面结合实施例具体介绍本发明。  The invention will be specifically described below in conjunction with the examples.
用移液管取 0.1NHC1溶液 50ml置于锥形瓶中， 加入干燥至恒重的盐酸阿霉 素粉末 10mg， 搅拌使其溶解， 再用微量移液器加入1.5%06^^117。0.31111，磁力 搅拌器作用下， 缓慢加入 α—氰基丙烯酸正丁酯 0.2ml， 边滴加边搅拌， 控制 在 10分钟加完。 封闭瓶口， 持续搅拌 1000rpm3h， 待出现橙黄色乳光， 用 0.2N NaOH溶液调节 PH=7， 过 0.45μπι滤膜。 滤液于真空干燥箱中 25°C、 0. lkPa条件 下干燥， 即制得本发明。 Pipette 50 ml of 0.1NHC1 solution into a conical flask, add 10 mg of doxorubicin hydrochloride powder dried to constant weight, stir to dissolve, and then add 1.5% 06^^11 7 with a micropipette. 0.31111, under the action of a magnetic stirrer, 0.2 ml of n-butyl α-cyanoacrylate was slowly added, and stirred while stirring, and the addition was completed in 10 minutes. Close the mouth of the bottle, continue to stir at 1000 rpm for 3 h, wait for orange-yellow milk to light, adjust pH = 7 with 0.2N NaOH solution, and pass 0.45 μm filter. The filtrate was dried in a vacuum oven at 25 ° C under a pressure of 0.1 kPa to obtain the present invention.

Claims

权 利 要 求 Rights request
1、 一种阿霉素一聚氰基丙烯酸正丁酯纳米粒的制备方法， 其步骤是： 用 HC1溶液与盐酸阿霉素粉末作用， 搅拌使其溶解， 再加入 Dextran™, 加入 α— 氰基丙烯酸正丁酯， 搅拌待出现橙黄色乳光， 调节 ΡΗ=7， 过 0.45μπι滤膜， 干 燥， 即制得本发明。  A method for preparing doxorubicin-n-butyl cyanoacrylate nanoparticles, the steps of which are: using HCl solution and doxorubicin hydrochloride powder, stirring to dissolve, adding DextranTM, adding α-cyanide The present invention was prepared by stirring n-butyl acrylate, stirring to be orange-yellow milk, adjusting ΡΗ=7, passing through a 0.45 μm filter, and drying.
2、 按权利要求 1所述的阿霉素一聚氰基丙烯酸正丁酯纳米粒的制备方法， 设制取一份 D0X— PBCA— ΝΡ， 其步骤是： 用移液管取 0.1N HC1溶液 50ml， 置于 锥形瓶中， 加入干燥至恒重的盐酸阿霉素粉末 10mg， 搅拌使其溶解， 再用微量 移液器加入1.5%0^1^&117。0.31111，磁力搅拌器作用下，缓慢加入 α—氰基丙烯 酸正丁酯 0.2ml， 边滴加边搅拌， 控制在 10分钟加完； 封闭瓶口， 持续搅拌 lOOOrpm 3h, 待出现橙黄色乳光， 用 0.2NNaOH溶液调节 PH二 7， 过 0.45μπι滤膜， 滤液于真空干燥箱中 25°C、 一 O.lkPa条件下干燥， 即制得本发明。 2. The method for preparing doxorubicin-n-butyl cyanoacrylate nanoparticles according to claim 1, wherein a D0X-PBCA- hydrazine is prepared, the steps of which are: using a pipette to take a 0.1 N HCl solution. 50 ml, placed in an Erlenmeyer flask, add 10 mg of doxorubicin hydrochloride powder dried to constant weight, stir to dissolve, and then add 1.5% 0^1^ & 11 7 with a micropipette. 0.31111, under the action of magnetic stirrer, slowly add 0.2ml of n-butyl α-cyanoacrylate, stir while adding dropwise, control to add in 10 minutes; close the bottle mouth, continue to stir lOOOOrpm 3h, until orange yellow milk, The pH was adjusted by a 0.2 N NaOH solution, passed through a 0.45 μm filter, and the filtrate was dried in a vacuum oven at 25 ° C under a 0.1 kPa condition to obtain the present invention.
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